不同部位喉黏膜间充质细胞的分离培养

目的：探讨不同部位喉黏膜间充质细胞的分离、培养方法,为喉部组织工程提供更多的可供选择的种子细胞。方法：收集临床上喉部手术病人切下的不同部位的喉黏膜,主要是会厌的背侧黏膜和声带的黏膜,各3例,共计6例。对会厌背侧的黏膜采用消化培养的方法,对声带部位的黏膜采用组织块培养法。最后通过免疫荧光染色对两种方法所获得的细胞进行鉴定,确定其来源于喉黏膜的间充质。结果：通过两种方法均可以成功获得相应部位的细胞,免疫荧光染色vimentin均呈阳性表达且CK均呈阴性表达,证明了获得的细胞确实是来源于间充质。结论：本实验成功的培养出了喉部不同部位的间充质细胞,为喉部的组织工程提供了更多的可供选择的种子细胞。     

比格犬会厌黏膜来源干细胞的分离培养与生物学特性鉴定

目的:探讨建立喉部黏膜间充质干细胞的分离培养方法,并对其生物学特性进行鉴定,为进一步研究其在喉部瘢痕中形成的作用及喉部组织工程提供参考.方法:以比格犬喉部会厌背侧(舌面)黏膜为研究对象,采用消化培养的方法分离具有间充质干细胞样细胞.选取第3代细胞对其进行生物学特性鉴定,首先利用MTT法检测其增殖活性及克隆形成情况,然后通过流式细胞术检测细胞表面分子标记物CD29及CD34的表达情况,最后应用第3代细胞对其进行成脂肪细胞和成骨细胞分化培养,观察其分化能力.结果:分离培养细胞形态较为一致,绝大多数呈梭形,排列不规则.MTT增殖活性实验及克隆形成试验结果显示,所分离的细胞具有良好的增殖活性和克隆形成率;流式细胞术结果显示,该细胞表达CD29间充质干细胞细胞表面标记物,低表达造血干细胞细胞表面标记物CD34;同时,该细胞诱导分化成脂肪细胞和成骨细胞实验表明,其具有多向分化潜能.结论:从比格犬会厌背侧黏膜分离的细胞具有间充质干细胞样特性,为进一步研究喉部瘢痕形成机制及喉部组织工程提供了技术基础.     

鼻黏膜间充质干细胞的培养、鉴定及诱导分化

目的建立Sprague Dawley(SD)大鼠鼻黏膜间充质干细胞(NM-MSCs)的获取、分离、培养方法,初步了解其生物学特性。方法通过SD大鼠鼻黏膜贴壁法体外分离、培养NM-MSCs,光镜下细胞形态学观察,用免疫荧光技术检测间充质干细胞和神经干细胞标记物,用流式细胞术检测细胞表面标记物,再诱导其向骨组织及脂肪组织方向分化,最后对其进行细胞周期检测及分析。结果分离培养的NM-MSCs光镜下以梭形与多角形细胞为主,呈放射状排列,且生长旺盛;免疫荧光染色显示NM-MSCs同时表达STRO-1和Nestin;第4代NM-MSCs不表达CD19、CD31、CD34、CD45及HLADR细胞表面标记物,但表达CD90、CD105基质细胞标记物;NM-MSCs经成骨、成脂诱导后,茜素红染色和油红O染色均呈阳性;细胞周期分析显示NM-MSCs符合干细胞生长的特性。结论 SD大鼠NM-MSCs组织块贴壁法获取容易,操作简单,且可大量扩增用于细胞实验研究,经体外诱导后具有多向分化潜能,具有间充质干细胞的一般生物学特性。SD大鼠鼻黏膜组织块贴壁法为组织细胞工程研究提供了充足的种子细胞来源。     

人羊膜间充质细胞分离培养及其干细胞特性研究

目的:研究人羊膜间充质细胞(Humanamnioticmesenchymalcells,HAMCs)的分离、培养及其干细胞特性,为羊膜间充质细胞在再生医学的潜在应用奠定实验基础。方法:无菌条件下取正常足月剖腹产胎儿的羊膜剪成碎片,经胰酶胶原酶序贯消化,DMEM/F12培养,倒置显微镜下观察其形态,MTT法检测其生长规律,免疫荧光的方法对细胞进行鉴定,定向诱导方法检测细胞的多向分化潜能。结果:来源于羊膜的间充质细胞,细胞免疫荧光显示SSEA-4,OCT-4阳性,具有很强的增殖能力,并且具有一定的多向分化能力,在特定条件下可分化为脂肪细胞和成骨细胞;结论:羊膜间充质细胞能够在体外分离、培养、扩增,并且具有干细胞特性。羊膜间充质细胞在再生医学和组织工程应用有很好的前景。     

外胚间充质干细胞构建组织工程骨骼肌的应用研究

目的:探讨利用大鼠颌突外胚间充质干细胞构建组织工程骨骼肌的可行性,并观察对骨骼肌缺损的修复重建的促进效应。方法:取妊娠E 11.5胎鼠颌突外胚间充质干细胞,纯化后在含5ml/L体积浓度二甲基亚砜的DMEM/F12培养基中诱导分化为骨骼肌样细胞,将细胞种植于BAM膜上培养形成组织工程骨骼肌。将其移植入大鼠骨骼肌缺损模型,手术后14 d观察骨骼肌恢复情况,同期进行组织学及免疫组化染色鉴定。结果:经诱导后外胚间充质干细胞可向骨骼肌样细胞转化,构建的组织工程骨骼肌可加速缺损的修复重建,组织学染色显示外胚间充质干细胞具有正常骨骼肌的组织形态,可表达成肌相关蛋白MyOD。结论:诱导后的外胚间充质干细胞可作为种子细胞构建组织工程骨骼肌,本实验为临床肌肉的缺损修复奠定了理论基础。     

3种间充质干细胞成软骨分化潜能比较

目的:比较骨髓间充质干细胞、脂肪间充质干细胞、滑膜间充质干细胞3种间充质干细胞的成软骨分化潜能,为软骨组织工程中种子细胞的选择提供实验依据。方法:采用贴壁法分别分离提取兔骨髓间充质干细胞、脂肪间充质干细胞、滑膜间充质干细胞3种间充质干细胞,并进行传代培养,绘制3种间充质干细胞的生长曲线并比较其倍增时间。将3种间充质干细胞成软骨诱导14 d后,行甲苯胺蓝染色及II型胶原免疫组化染色以观测3种细胞成软骨分化能力。结果:脂肪间充质干细胞的倍增时间短于骨髓间充质干细胞,滑膜间充质干细胞的倍增时间最短;3种细胞成软骨诱导14 d后均产生糖胺聚糖和II型胶原,且组与组之间II型胶原表达水平的差异有统计学意义,骨髓间充质干细胞组高于脂肪间充质干细胞组(P0.01),滑膜间充质干细胞组高于骨髓间充质干细胞组(P0.01)。结论:在一定的培养条件下,3种间充质干细胞均有一定的成软骨细胞分化潜能,滑膜间充质干细胞最快的增殖速度及最强的成软骨分化潜能。     

人胎盘底蜕膜间充质干细胞库的初步构建

该文探讨了建立生物学特性稳定的人胎盘底蜕膜间充质干细胞(DB-MSCs)库的可行性,旨在为组织工程种子细胞提供更多来源。该研究采用组织块贴壁法从10例足月人胎盘底蜕膜组织中获取间充质干细胞,并采用STR检测细胞是否均来源于母体组织。对分离得到的DB-MSCs采用–196 °C低温冻存,并在一定时间复苏培养,用倒置显微镜观察细胞形态,CCK8法检测细胞增殖能力,流式细胞仪分析细胞周期和细胞表面标志物,特定培养基诱导其成脂、成骨、成软骨分化。结果显示,从底蜕膜组织中分离培养扩增获得数目稳定的DB-MSCs。STR分析证明,所得细胞均来源于母体组织,经冻存并复苏后的DB-MSCs形态和表面标志物保持不变,并可稳定扩增8代以上,倍增时间为(2.04±0.25)天,大多数的细胞处于静止期(G_0/G_1),复苏后的细胞保持了较强的成脂、成骨和成软骨分化能力,在细胞中未发现细菌、真菌、支原体和内毒素等的污染。该研究初步建立了DB-MSCs库,进一步明确了库存细胞的制备流程及其质量评价体系,可为再生医学修复重建组织工程提供较好的种子细胞。     

人羊膜间充质细胞分离培养及其干细胞特性研究

目的：研究人羊膜间充质细胞（Humanamnioticmesenchymalcells,HAMCs）的分离、培养及其干细胞特性,为羊膜间充质细胞在再生医学的潜在应用奠定实验基础。方法：无菌条件下取正常足月剖腹产胎儿的羊膜剪成碎片,经胰酶胶原酶序贯消化,DMEM/F12培养,倒置显微镜下观察其形态,MTT法检测其生长规律,免疫荧光的方法对细胞进行鉴定,定向诱导方法检测细胞的多向分化潜能。结果：来源于羊膜的间充质细胞,细胞免疫荧光显示SSEA-4,OCT-4阳性,具有很强的增殖能力,并且具有一定的多向分化能力,在特定条件下可分化为脂肪细胞和成骨细胞;结论：羊膜间充质细胞能够在体外分离、培养、扩增,并且具有干细胞特性。羊膜间充质细胞在再生医学和组织工程应用有很好的前景。     

脐带间充质干细胞诱导分化为类许旺细胞的实验研究

目的:探讨通过化学诱导的方法,诱导脐带间充质干细胞分化为类许旺细胞,为组织工程神经寻找一种新的种子细胞来源。方法:通过酶消化法,分离获得原代脐带间充质干细胞。体外培养至第3代(P3),以1×103/cm2接种于培养瓶中,待细胞长至亚融合状态,吸弃培养液,加入含β-巯基乙醇的培养液预诱导24 h。然后,加入含有全反式维甲酸的培养液进一步诱导72 h。最后,加入含有胶质细胞生长因子的培养液,作用两周。对诱导后的脐带间充质干细胞用免疫荧光及RT-PCR法,进行形态观察和表型鉴定。结果:经过上述诱导过程,脐带间充质干细胞的形态,由开始时的扁平、片状、多极,类似成纤维细胞状,逐渐变为长梭形,双极或三极,类似许旺细胞。同时,表达许旺细胞特异性标志S-100、P75;而且S100,P75的m RNA水平也明显上调,并且可以观测到GFAP的m RNA条带。结论:化学诱导法,可以将脐带间充质干细胞诱导分化为类许旺细胞,有望为组织工程神经提供一种新的许旺细胞来源。     

兔骨髓间充质干细胞的体外分离培养和生物学特性观察

目的:探讨兔骨髓间充质干细胞体外分离、培养和鉴定方法,观察其生物学特性.方法:采集兔股骨及胫骨骨髓组织,采用密度梯度离心法结合贴壁培养法体外分离、培养和扩增兔骨髓间充质干细胞,倒置相差显微镜观察细胞形态,绘制原代、第1、3、8代细胞生长曲线,流式细胞术检测细胞表面标志物,成骨和成脂肪诱导培养鉴定,观察细胞生物学特性.结果:培养的BMSCs呈纺锤形、长梭形,旋涡状排列、放射性生长,增值活跃.各代细胞生长曲线呈S型,细胞增值活跃.细胞表面标志物CD44分子阳性,CD34和CD45分子阴性.经成骨和成脂肪诱导后细胞碱性磷酸酶染色和油红O染色阳性.结论:成功建立了兔BMSCs体外分离、培养的有效方法,扩增的BMSCs仍保留多向分化潜能,是理想的组织工程种子细胞.     

Mechanotransduction of vocal fold fibroblasts and mesenchymal stromal cells in the context of the vocal fold mechanome

The design of cell-based therapies for vocal fold tissue engineering requires an understanding of how cells adapt to the dynamic mechanical forces found in the larynx. Our objective was to compare mechanotransductive processes in therapeutic cell candidates (mesenchymal stromal cells from adipose tissue and bone marrow, AT-MSC and BM-MSC) to native cells (vocal fold fibroblasts-VFF) in the context of vibratory strain. A bioreactor was used to expose VFF, AT-MSC, and BM-MSC to axial tensile strain and vibration at human physiological levels. Microarray, an empirical Bayes statistical approach, and geneset enrichment analysis were used to identify significant mechanotransductive pathways associated with the three cell types and three mechanical conditions. Two databases (Gene Ontology, Kyoto Encyclopedia of Genes and Genomes) were used for enrichment analyses. VFF shared more mechanotransductive pathways with BM-MSC than with AT-MSC. Gene expression that appeared to distinguish the vibratory strain condition from polystyrene condition for these two cells types related to integrin activation, focal adhesions, and lamellipodia activity, suggesting that vibratory strain may be associated with cytoarchitectural rearrangement, cell reorientation, and extracellular matrix remodeling. In response to vibration and tensile stress, BM-MSC better mimicked VFF mechanotransduction than AT-MSC, providing support for the consideration of BM-MSC as a cell therapy for vocal fold tissue engineering. Future research is needed to better understand the sorts of physical adaptations that are afforded to vocal fold tissue as a result of focal adhesions, integrins, and lamellipodia, and how these adaptations could be exploited for tissue engineering.     

Vocal fold composition and early glottic carcinoma infiltration

ABSTRACT: BACKGROUND: Current imaging techniques provide only limited information pertaining to the extent of infiltration of laryngeal carcinomas into vocal fold tissue layers. Therefore, it is needed to seek the contribute to the body of knowledge surrounding examination and characterization in laryngeal carcinoma infiltration. METHODS: Excised larynges were collected from 30 male laryngectomy patients with an average age of 43.5 years (ranging 36 to 55 years) and history of smoking ([GREATER-THAN OR EQUAL TO]10 years) exhibiting T1, T2, or subglottal (normal vocal fold) carcinomas. Vocal folds were preserved via freezing or immersion in paraffin. The depth of the mucosa, submucosa, and muscular layers in both normal vocal folds and tumor tissues of afflicted vocal folds was measured. RESULTS: The average depths of the mucosa, submucosa, and muscular layers in normal vocal folds were 0.15 [PLUS-MINUS SIGN] 0.06 mm, 2.30 [PLUS-MINUS SIGN] 0.59 mm, and 2.87 [PLUS-MINUS SIGN] 0.88 mm, respectively. Infiltration measurements of T1 tumors showed a depth of 1.62 [PLUS-MINUS SIGN] 0.51 mm and 1.32 [PLUS-MINUS SIGN] 0.49 mm in frozen sections and paraffin-embedded samples, respectively. Similarly, T2 tumors showed a depth of 2.87 [PLUS-MINUS SIGN] 0.68 mm and 2.58 [PLUS-MINUS SIGN] 0.67 mm in frozen sections and paraffin-embedded samples, respectively. T1 and T2 tumors occupied 24.8 [PLUS-MINUS SIGN] 10 and 48.5 [PLUS-MINUS SIGN] 15 percent of the normal vocal fold depth, respectively. CONCLUSION: This data provides a baseline for estimating infiltration of laryngeal carcinomas in vocal fold tissue layers, of particular interest to surgeons. This information may be used to assess typical depths of infiltration, thus allowing for more appropriate selection of surgical procedures based on individual patient assessment.     

Regeneration of Vocal Fold Mucosa Using Tissue-Engineered Structures with Oral Mucosal Cells

Objectives

Scarred vocal folds result in irregular vibrations during phonation due to stiffness of the vocal fold mucosa. To date, a completely satisfactory corrective procedure has yet to be achieved. We hypothesize that a potential treatment option for this disease is to replace scarred vocal folds with organotypic mucosa. The purpose of this study is to regenerate vocal fold mucosa using a tissue-engineered structure with autologous oral mucosal cells.

Study Design

Animal experiment using eight beagles (including three controls).

Methods

A 3 mm by 3 mm specimen of canine oral mucosa was surgically excised and divided into epithelial and subepithelial tissues. Epithelial cells and fibroblasts were isolated and cultured separately. The proliferated epithelial cells were co-cultured on oriented collagen gels containing the proliferated fibroblasts for an additional two weeks. The organotypic cultured tissues were transplanted to the mucosa-deficient vocal folds. Two months after transplantation, vocal fold vibrations and morphological characteristics were observed.

Results

A tissue-engineered vocal fold mucosa, consisting of stratified epithelium and lamina propria, was successfully fabricated to closely resemble the normal layered vocal fold mucosa. Laryngeal stroboscopy revealed regular but slightly small mucosal waves at the transplanted site. Immunohistochemically, stratified epithelium expressed cytokeratin, and the distributed cells in the lamina propria expressed vimentin. Elastic Van Gieson staining revealed a decreased number of elastic fibers in the lamina propria of the transplanted site.

Conclusion

The fabricated mucosa with autologous oral mucosal cells successfully restored the vocal fold mucosa. This reconstruction technique could offer substantial clinical advantages for treating intractable diseases such as scarring of the vocal folds.     

Different patterns of cytokeratin expression in the normal epithelia of the upper respiratory tract

The distribution and type of cytokeratins present in the normal human epithelia of the nasopharynx, oropharynx, tongue, palatine tonsil, epiglottis, vocal cord, and laryngeal ventricle were studied using immunohistochemical techniques and by gel electrophoresis of cytoskeletal proteins microdissected from frozen tissues. Noncornifying stratified epithelia covering the oropharynx, tongue, surface of the palatine tonsil, pharyngeal surface of the epiglottis, and vocal cord were all found to contain cytokeratins nos. 4, 5, 6, 13, 14, and 15, together with minor amounts of cytokeratin no. 19, i.e., a pattern similar to that previously reported for esophageal epithelium. The immunohistochemical reaction with KA4, an antibody specific for cytokeratins nos. 14, 15, 16, and 19, revealed reactivity confined to the basal epithelial cells of the tongue, oropharynx, pharyngeal epiglottis, and two out of five samples of vocal cords. This same antibody reacted with the entire thickness of three out of the five true vocal cords which were shown by gel electrophoresis to also contain cytokeratins nos. 16 and 17. Gel electrophoresis revealed that the pseudostratified columnar epithelium covering the laryngeal ventricle was more complex, in that it contained cytokeratins nos. 5, 13, 14, 15, and 17, which are typical of stratified epithelia, as well as cytokeratins nos. 7, 8, 18, and 19, which are characteristic of simple epithelia. This pattern is similar to that found in bronchial epithelium. The laryngeal surface of the epiglottis exhibited cytokeratins nos. 4, 5, 7, 8, 13, 14, 15, 17, 18, and 19, i.e., a pattern combining features of both esophageal- and bronchial-type epithelia. The reaction of these epithelia containing columnar cells with antibody RGE-53, which is specific for cytokeratin no. 18, revealed a staining reaction confined to the superficial columnar cells, whereas KA1 stained only the basal cells of these epithelia. The results of our study make it possible to distinguish two types of noncornifying stratified squamous epithelium, namely the 'esophageal type' which covers the tongue, oropharynx, and pharyngeal surface of the epiglottis, and another type which overlies the vocal cords and the transitional zone between the pharyngeal and laryngeal surfaces of the epiglottis. Furthermore, there appear to be variants of pseudostratified columnar epithelium, i.e., the usual bronchial type lining the laryngeal ventricle, and a type with a thicker subcolumnar cell compartment that is found on the laryngeal surface of the epiglottis. The patterns of expression of cytokeratins in the respiratory tract are compared with those of other epithelia.     

喉癌中SP100 表达及其与临床病理关系

目的：通过对不同喉癌病人癌组织SP100蛋白进行测量,明确SP100 在喉癌的发生发展过程中的作用并探究其与临床病理 的关系。方法：通过对喉癌手术病人切除的癌组织和癌周组织进行固定、脱水、包埋、切片、免疫组织化学染色判断SP100 阳性细 胞个数以及探究其与临床病理学之间的关系。结果：在正常黏膜上皮细胞和分化良好的癌细胞中,以细胞核内染色为主,在低分 化癌细胞中,在细胞质内呈弥漫性分布。SP100 蛋白在癌旁正常黏膜上皮组织中表达的阳性率比喉癌原发灶中高。SP100 蛋白在 96 例喉癌组织中的表达水平与病理分化程度密切相关(P<0.05 ),而与患者性别、年龄、P-TNM 分期、淋巴结转移无相关性 (P>0.05)。结论：喉癌组织中SP100蛋白表达水平、细胞内分布状况在不同分化程度癌细提示在喉癌的不同阶段可能发挥不同的 作用。     

Morphological aspects of the euphonic voice

The high quality of a euphonic voice is the result of complex interactions between many organs and systems. Vibrating vocal folds play a crucial role in this process. Their physiological motion is conditioned by the presence of the layered structure of laryngeal mucosa. In this study, we assessed the degree of dysphonia according to the Union of European Phoniatrics (UEP) scale. Videoendoscopy (VLS) and videostroboscopic (VLSS) examination of the larynx was used to visualize the vibration of the vocal folds. Morphological assessment of the inter-membranous part of the vocal fold mucosa was carried out using material collected after surgical treatment (60%) or obtained from autopsy (40%). The samples were examined by light microscopy and transmission electron microscopy. In euphonic voices, 1° of dysphonia (UEP) and the physiological endoscopic (VLS) and stroboscopic (VLSS) findings of vocal folds were registered. No morphological or ultramorphological changes were observed in the cells of the multilayered flat epithelium, basal membrane or in the stroma. Unchanged epithelial cells were situated on the basal membrane with folds. Moreover, numerous pericytes, vessels with multiplication of basal membranes, scanty collagenous fibers, plasmatic cells and lymphocytes were seen. Morphological changes with signs of atrophy and polypoid degeneration of the vocal fold mucosa were found in only 3 (15%) patients.     

喉癌中SP100表达及其与临床病理关系

目的：通过对不同喉癌病人癌组织SP100蛋白进行测量,明确SP100在喉癌的发生发展过程中的作用并探究其与临床病理的关系。方法：通过对喉癌手术病人切除的癌组织和癌周组织进行固定、脱水、包埋、切片、免疫组织化学染色判断SP100阳性细胞个数以及探究其与临床病理学之间的关系。结果：在正常黏膜上皮细胞和分化良好的癌细胞中,以细胞核内染色为主,在低分化癌细胞中,在细胞质内呈弥漫性分布。SP100蛋白在癌旁正常黏膜上皮组织中表达的阳性率比喉癌原发灶中高。SP100蛋白在96例喉癌组织中的表达水平与病理分化程度密切相关（P〈0．05）,而与患者性别、年龄、P-TNM分期、淋巴结转移无相关性（P〉0．05）。结论：喉癌组织中SP100蛋白表达水平、细胞内分布状况在不同分化程度癌细提示在喉癌的不同阶段可能发挥不同的作用。     

声带组织切片方法的比较

目的:探讨犬声带冠状位切片与水平位切片各自的特点,为声带实验提供合适的切片方法。方法:家犬4只,2只取材后行冠状位石蜡切片,2只取材后行水平位石蜡切片。通过HE染色观察声带固有层的一般组织结构,Masson三色染色观察固有层中胶原的排列情况。结果:HE染色示冠状位、水平位切片均可见声带表面被覆复层鳞状上皮,固有层内有大量排列紧密的纤维组织,纤维组织中夹杂少量腺体,固有层下方为肌层。冠状位切片可观察声带某一点冠状面固有层的情况,若观察整个声带的情况需声带连续切片;水平位切片可在一张切片中观察到前联合、声带膜部及声带突部位的固有层情况,解剖标志明显,利于定位。Masson三色染色示冠状位、水平位切片均可见固有层浅层有较细的胶原纤维束,中层有较粗的纤维束与较细的纤维束交织排列,深层纤维束排列更紧密。结论:冠状位切片可观察声带某一点冠状面固有层的整体情况,水平位切片可在一张切片中观察到前联合、膜部及声带突部位的固有层情况。